Evaluation of muscle lipid extraction and non-lethal fin tissue use for carbon, nitrogen, and sulfur isotope analyses in adult salmonids.

RATIONALE: Chemical lipid extraction or using alternative tissues such as fish fin as opposed to muscle may alter isotopic ratios and influence interpretations of δ13 C, δ15 N, and previously unassessed δ34 S values in stable isotope analyses (SIA). Our objectives were to determine if lipid extraction alters these isotope ratios in muscle, if lipid normalization models can be used for lipid-rich salmonids, and if fin isotope ratios are comparable with those of muscle in adult salmonids. METHODS: In six adult salmonid species (n = 106) collected from Lake Ontario, we compared three isotope ratios in lipid-extracted (LE) muscle with bulk muscle, and LE muscle with fin tissue, with paired t-tests and linear regressions. We compared differences between δ13 C values in LE and bulk muscle with predicted values from lipid normalization models and the log-linear model of best fit and determined model efficiency. RESULTS: The δ15 N values in LE muscle increased (<1‰) relative to bulk muscle for most salmonids, with relationships nearing 1:1. There were either no differences or strong 1:1 relationships in δ34 S values between species-specific bulk and LE muscle. One lipid normalization model had greater model efficiency (97%) than the model of best fit (94%). Fin had higher δ13 C values than LE muscle while δ15 N trends varied (<1‰); however, both isotope ratios had either no or weak linear relationships with fin and LE muscle within species. The δ34 S values in fin were similar to those in LE muscle and had strong 1:1 relationships across species. CONCLUSIONS: We recommend using the lipid normalization model to adjust for δ13 C values in lipid-rich muscle (C:N >3.4). LE muscle could be used without δ15 N or δ34 S adjustments, but the minimal increase in δ15 N values may affect SIA interpretation. With high unexplained variability among adult species in fin-muscle δ13 C and δ15 N relationships, species-specific fin-muscle adjustments are warranted. No fin-muscle tissue adjustment would be required for δ34 S values.

Rapid NMR-scale purification of 15N,13C isotope-labeled recombinant human STIM1 coiled coil fragments.

We report a new NMR-scale purification procedure for two recombinant wild type fragments of the stromal interaction molecule 1 (STIM1). This protein acts as a calcium sensor in the endoplasmic reticulum (ER) and extends into the cytosol accumulating at ER - plasma membrane (PM) junctions upon calcium store depletion ultimately leading to activation of the Orai/CRAC channel. The functionally relevant cytosolic part of STIM1 consists of three coiled coil domains, which are mainly involved in intra- and inter-molecular homomeric interactions as well as coupling to and gating of CRAC channels. The optimized one-step rapid purification procedure for two 15N,13C isotope-labeled cytosolic coiled coil fragments, which avoids the problems of previous approaches. The high yields of soluble well folded 15N,13C isotope-labeled cytosolic coiled coil fragments followed by detergent screening provide for initial NMR characterization of these domains. The longer 30.5 kDa fragment represents the largest STIM1 wild type fragment that has been recombinantly prepared and characterized in solution without need for mutation or refolding.

On the problem of resonance assignments in solid state NMR of uniformly ¹5N,¹³C-labeled proteins.

Determination of accurate resonance assignments from multidimensional chemical shift correlation spectra is one of the major problems in biomolecular solid state NMR, particularly for relative large proteins with less-than-ideal NMR linewidths. This article investigates the difficulty of resonance assignment, using a computational Monte Carlo/simulated annealing (MCSA) algorithm to search for assignments from artificial three-dimensional spectra that are constructed from the reported isotropic (15)N and (13)C chemical shifts of two proteins whose structures have been determined by solution NMR methods. The results demonstrate how assignment simulations can provide new insights into factors that affect the assignment process, which can then help guide the design of experimental strategies. Specifically, simulations are performed for the catalytic domain of SrtC (147 residues, primarily ß-sheet secondary structure) and the N-terminal domain of MLKL (166 residues, primarily α-helical secondary structure). Assuming unambiguous residue-type assignments and four ideal three-dimensional data sets (NCACX, NCOCX, CONCA, and CANCA), uncertainties in chemical shifts must be less than 0.4 ppm for assignments for SrtC to be unique, and less than 0.2 ppm for MLKL. Eliminating CANCA data has no significant effect, but additionally eliminating CONCA data leads to more stringent requirements for chemical shift precision. Introducing moderate ambiguities in residue-type assignments does not have a significant effect.

Isolation and stable nitrogen isotope analysis of ammonium ions in ammonium nitrate prills using sodium tetraphenylborate.

RATIONALE: Because of the threat of bombings using improvised explosives containing ammonium nitrate (AN), law enforcement and intelligence communities have been interested in stable isotope techniques for tracking and discriminating AN sources. Separate analysis of the AN component ions ammonium and nitrate would add discriminatory power to these techniques. METHODS: Ammonium ions in dissolved AN solution were isolated from samples by precipitation using sodium tetraphenylborate solution. We tested the isolation of ammonium from nitrates using solutions of ammonium and nitrate salts with different (15)N/(14)N isotope ratios. Ammonium tetraphenylborate and AN were separately analyzed for their (15)N/(14)N isotope ratios using EA-ConFlo-IRMS, and the (15)N/(14)N isotope ratios of the nitrate ions were calculated using mass balance. Ammonium and nitrate nitrogen isotope ratios were plotted as two separate variables. RESULTS: Isolation of ammonium precipitate from solutions containing dissolved nitrates did not influence the nitrogen isotope ratios of test ammonium salts. A survey set of 42 AN samples showed that the ammonium and nitrate (15)N/(14)N isotope ratios were not significantly correlated, and the paired mean differences were not statistically significant. Both ammonium and nitrate were depleted in (15)N relative to their theoretical atmospheric sources. CONCLUSIONS: Isolation of the ammonium ion from AN adds another dimension for the discrimination of forensic AN samples. This technique using sodium tetraphenylborate is robust and does not require specialized equipment. Our observations indicated that ammonium nitrogen and nitrate nitrogen have independent sources of isotopic variation.

Coupling a Knudsen reactor with the short lived radioactive tracer (13)N for atmospheric chemistry studies.

A Knudsen cell flow reactor was coupled to an online gas phase source of the short-lived radioactive tracer (13)N to study the adsorption of nitrogen oxides on ice at temperatures relevant for the upper troposphere. This novel approach has several benefits over the conventional coupling of a Knudsen cell with a mass spectrometer. Experiments at lower partial pressures close to atmospheric conditions are possible. The uptake to the substrate is a direct observable of the experiment. Operation of the experiment in continuous or pulse mode allows to retrieve steady state uptake kinetics and more details of adsorption and desorption kinetics.

Dynamic modelling and simulation of ¹5N separation by chemical exchange in NO, NO2-HNO3 system.

The dynamic behaviour of a ¹5N separation process by chemical exchange in a NO, NO2-HNO3 system has been analysed based on an accurate mathematical model. A nonlinear system of first-order partial differential equations was determined by considering the multiple exchange reactions between the components of the gaseous mixture and the liquid phase constituents. The mathematical model of the process describes the space-time variation of the ¹5N mole fraction in gas and liquid phases and provides a better understanding of operating limits and decision support in process design and optimisation.

Isolation and desalting with cation-exchange chromatography for compound-specific nitrogen isotope analysis of amino acids: application to biogeochemical samples.

We have established a procedure for removing interfering materials from extracts of geological and biological samples, in order to determine precise compound-specific nitrogen isotopic compositions of amino acids. We employed cation-exchange chromatography of protein and non-protein amino acids prior to derivatization for gas chromatographic separation. The average recovery of a standard amino acid solution was better than 94%, without nitrogen isotope fractionation during the cation-exchange chromatography. We applied the procedure to various environmental samples including 'hard' (calcareous, siliceous, rock and sediment samples) and 'soft' materials (aggregated microbial samples and biological soft tissue samples). We conclude that cation-exchange chromatography is a pre-treatment procedure which should be widely useful for the determination of compound-specific nitrogen isotopic compositions of amino acids.

Experimental plant for simultaneous production of (14)N and (15)N by (15)N/(14)N exchange in NO, NO(2)-HNO(3) system under pressure.

An experimental study on (14)N and (15)N simultaneous separation using the chemical exchange in NO, NO(2)-HNO(3) system under pressure is presented. The influence of the pressure and of the interstage 10 M HNO(3) flow rate on the separation of (14)N and (15)N was measured on a packed column with product and waste refluxers. At steady state and 1.8 atm (absolute), the isotopic concentration at the bottom of the separation column was 0.563 at% (15)N, and in the top of the column was 0.159 at% (15)N. The height equivalent to a theoretical plate and interstage 10 M HNO(3) flow rate values, obtained in these experimental conditions, allows the separation of (14)N highly depleted of (15)N and of (15)N at 99 at% (15)N concentration.